Team:UNITN-Trento/Notebook/Labposts/07/03

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Revision as of 08:21, 3 October 2013

{ "date" : "2013-07-02", "author" : "gabriele", "title" : "Another busy day", "content" : "

SAM synthetase ligation in (linear) pSB1C3

First of all, early in the morning I added 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°C).

Then, I purified and quantified the two digestion samples:

Sample	Quantity
G1 SAMsynthetase	18.8ng/µl
G2 linear pSB1C3	6.5ng/µl

After that, I performed the ligation (the plasmid had a too low concentration).

	Ctrl	0.5:1	0.5:2
Buffer	3µl
Plasmid	14.3µl
Insert	0	6µl	12µl
Ligase	1µl
Water	11.7µl	5.7µl	0µl

And, \"finally\", I transformed the ligations in NEB10β cells.

(2) SAMsynthetase amplification

I amplified SAMsynthetase from the E2 sample (that was purified yesterday) following the usual protocol. The PCR was performed in triplicates.

The PCR products were run on a 1% agarose gel.

Gel

Loading scheme
E1A	E1B	E1C	empty	1kb ladder

$\"Gel\"$

As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.

(3) R0010 amplification

I also amplified R0010 from A sample (R0010, 243.7ng/µl) from 27/06. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86°C) and the suffix Rv (Tm = 90°C).

PCR Mixes

	Mix HF (x3)	Mix GC (x2)
Phusion GC buffer	0	10µl
Phusion HF buffer	10µl	0
dNTPs	1µl
primer Fw	2.5µl
primer Rv	2.5µl
template (50ng/µl)	0.5µl
Phusion pol	0.5µl
Water	33µl

Given that R0010 is 200bp long, the PCR program was the following:

PCR settings

PCR setting
Step	Temperature	Time	Go to
1	98°C	30 sec
2	98°C	10 sec
3	72°C	3 sec	step #2, 30 times
4	72°C	10 min
5	4°C	pause

Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.

Gel

Loading scheme
100bp ladder
AHF1
AHF2
AHF3
AGC1
AGC2
empty
1kb ladder

$\"Gel\"$

As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!

(4) linear pSB1C3 amplification

I didn't know that linear pSB1C3 is \"impossible\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.

Gel

Loading scheme
1kb ladder	G3A	G3B	G3C

$\"Gel\"$

", "tags" : "SAMsynthetase-Plac-pSB1C3" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-07-18",
+	"date" : "2013-07-02",
-	"author" : "emil",
+	"author" : "gabriele",
-	"title" : "The incompetent Bacillus subtilis saga medium episode ",
+	"title" : "Another busy day",
-	"content" : "<html>I have prepared a minimal medium for bacillus following the Cambridge Igem 2012 protocol and I did an inocula of bacillus from a glicerol stock 50% for the following day.Moreover I tried to grow some competent NEB10&beta; following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-prep\"> competent cell protocol </a>.</html>",
+	"content" : "<html><h3>SAM synthetase ligation in (linear) pSB1C3</h3>First of all, early in the morning I added 1&micro;l of DpnI to the SAMsynthetase#G2 O/N digestion and 1&micro;l of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37&deg;C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80&deg;C).<br/><br/>Then, I purified and quantified the two digestion samples:<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1 <i>SAMsynthetase</i></td><td>18.8ng/&micro;l</td></tr><tr><td>G2 <i>linear pSB1C3</i></td><td>6.5ng/&micro;l</td></tr></table></center><br/>After that, I performed the ligation (the plasmid had a too low concentration).<center><table class=\"tn-sp-table\"><tr><td style=\"border:none\"></td><th>Ctrl</th><th>0.5:1</th><th>0.5:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">3&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.3&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>6&micro;l</td><td>12&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>11.7&micro;l</td><td>5.7&micro;l</td><td>0&micro;l</td></tr></table></center><br/>And, \"finally\", I transformed the ligations in NEB10&beta; cells.<br/><br/><hr><h3>(2) SAMsynthetase amplification</h3>I amplified SAMsynthetase from the E2 sample (that was purified <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">yesterday</a>) following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">usual protocol</a>. The PCR was performed in triplicates.<br/><br/>The PCR products were run on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"5\">Loading scheme</th></tr><tr><td>E1A</td><td>E1B</td><td>E1C</td><td><i>empty</i></td><td>1kb ladder</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.<br/><br/><hr><h3>(3) R0010 amplification</h3>I also amplified R0010 from <b>A</b> sample (R0010, 243.7ng/&micro;l) from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-27-gabriele-emil\">27/06</a>. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86&deg;C) and the suffix Rv (Tm = 90&deg;C).<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Mix HF (x3)</th><th>Mix GC (x2)</th></tr><tr><td>Phusion GC buffer</td><td>0</td><td>10&micro;l</td></tr><tr><td>Phusion HF buffer</td><td>10&micro;l</td><td>0</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>primer Fw</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>primer Rv</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>template (50ng/&micro;l)</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Water</td><td colspan=\"2\">33&micro;l</td></tr></table></center></html>}}<html><br/>Given that R0010 is 200bp long, the PCR program was the following:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR settings|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR setting</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>98&deg;C</td><td>30 sec</td><td></td></tr><tr><td>2</td><td>98&deg;C</td><td>10 sec</td><td></td></tr><tr><td>3</td><td>72&deg;C</td><td>3 sec</td><td>step #2, 30 times</td></tr><tr><td>4</td><td>72&deg;C</td><td>10 min</td><td></td></tr><tr><td>5</td><td>4&deg;C</td><td>pause</td><td></td></tr></table></center></html>}}<html><br/>Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>100bp ladder</td></tr><tr><td>AHF1</td></tr><tr><td>AHF2</td></tr><tr><td>AHF3</td></tr><tr><td>AGC1</td></tr><tr><td>AGC2</td></tr><tr><td><i>empty</i></td><tr><td>1kb ladder</td></tr></tr></table><img src=\"https://static.igem.org/mediawiki/2013/3/36/Tn-20130702-GG_R0010.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!<br/><br/><hr><h3>(4) linear pSB1C3 amplification</h3>I didn't know that linear pSB1C3 is \"<i>impossible</i>\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G3A</td><td>G3B</td><td>G3C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html></html>",
-	"tags" : "Bacillus_subtilis"
+	"tags" : "SAMsynthetase-Plac-pSB1C3"
 }